Regulation of DNA replication is one of the central events in the cell cycle of eukaryotic cells. We are studying the mechanisms and regulation of Bovine papilloma virus type 1 (BPV) replication, because latent viruses that replicate as nuclear plasmids may provide model systems to analyze replication control. It is our goal to understand the interplay between positive and negative cis-regulatory elements and trans-acting factors which are involved in the initiation of BPV DNA replication and which prevent re-replication within a single S-phase. Our approach will combine in vivo and in vitro studies. We will express viral replication genes in a heterologous mammalian expression system. The biochemical activities of the purified proteins will be assayed. We will further develop a cell free in vitro system which replicates BPV containing the origin of replication. A cell free replication system will allow us to determine the initiation sites of DNA synthesis and will permit the elucidation of the role of the positive and negative viral replication genes. We plan to create BPV mutants containing temperature-sensitive alleles of the viral replication genes. The genetic analysis of these mutants will permit the examination of the temporal requirements of the viral gene products involved in latent replication. The knowledge derived from studies of BPV should facilitate the characterization of cellular and viral components that permit extrachromosomal maintenance of human papilloma viruses in certain cells and that lead to integration of the viral genomes into the host cell genome in carcinoma cells.